Production of oxidases by aspergilli for monohydroxylation of steroids



United States Patent PRODUCTION OF OXIDASES BY ASPERGILLI FOR MONOHYDROXYLATION OF STEROIDS Eugene L. Dulaney, Rahway, N.J., assignor to Merck & Co., Inc., Rahway, NJ., a corporation of New Jersey No Drawing. Application January 6, 1955 Serial No. 480,304

8 Claims. (Cl. 195-67) This invention relates to methods for effecting the monohydroxylation of steroids by the action of microorganisms. More particularly, it is concerned with processes for the production of ll-hydroxy steroids by subjecting ll-desoxy steroids to the action of hydroxylating strains of micro organisms or the hydroxylating enzymes of such microorganisms under conditions which will inhibit the formation of polyhydroxy compounds.

(1' he discovery of the remarkable therapeutic properties of cortisone and similar related compounds having an oxygen substituent at 0-11 has stimulated wide interest in finding simpler and more economical methods of preparing such compounds. One of the principal difficulties encountered in the synthesis of cortisone and its related compounds is the introduction of the ll-oxygen substituent. Although various methods have been developed for the synthesis of ll-oxygenated steroids, such processes are not entirely satisfactory and other methods more suitable for the commercial preparation of ll-oxygen substituted steroids in high yields have been sought.

Methods for effecting the oxygenation of steroids by the action of microorganisms are known in the art. Thus, species of Actinomycetes are known to introduce oxygen into a number of positions of the steroid molecule, ineluding position 11. Similarly, various species of genera included in the order Mucorales also introduce oxygen in various positions of the steroid ring structure.

Unfortunately, the utilization of the known procedures for introducing oxygen substituents at position 11 by the action of microorganisms is beset by a number of troublesome diificulties. One such difiiculty is that the oxygenation results in the formation of a multiplicity of oxygenated products which are difficult to separate, thereby resulting in the obtainment of poor yields of the desired ll-oxygenated steroids. Thus, in the oxidation of steroids having methylene substituents at position 6 and 11 by the action of strains of Aspergillus, there is obtained a mixture of the ll-hydroxy steroid and the 6,11-dihydroxy steroid. The hydroxylation of the steroid in the 6 position in addition to the 11 position leads to the formation of an undesirable by-product which is difficult to separate from the desired ll-hydroxy compound and results in the obtainment of the desired compound in low yields. Accordingly, it would be very desirable indeed to provide a means which would prevent the undesired polyhydroxylation of steroids by the action of hydroxylating strains of microorganisms or their enzymes.

One object of the present invention is to provide a means for preventing or inhibiting the polyhydroxylation Aspergillus ochraceus.

droxylation of the steroids present.

growth of the microorganisms.

50 compound can be inhibited 5 jected to the action of hydroxylating strains of Aspergillus.

A further object is to provide a means for the conversion of progesterone to lla-hydroxy progesterone substantially free of 6(3,11u-dihydroxy progesterones by the action of enzymes produced by hydroxylating strains of These and other objects of my invention will be readily apparent from the detailed description of my invention hereinafter provided.

In accordance with my invention, it is now found that the polyhydroxylation by microorganisms can be in- 15 hibited or prevented under conditions whereby the growth of the microorganism is carefully controlled by the medium constituents. Under such conditions the enzyme system can be altered from that usually occurring to produce a system preventing or inhibiting the-polyhy- I have found that these carefully controlled conditions can be brought about either by limiting the amount of a specific element essential to the growth of the microorganisms, or by in- 'cluding in the medium a substance which will inhibit the By growing the microorganism under such conditions there is produced a system which will effect the polyhydroxylating ability of the microorganisms. The conditions under which a particular microorganism will permit monohydroxylation of steroids with a minimum amount of the undesired polyhydroxylated steroids can be determined by a series of test fermentations of the organism and the steroid to be oxygenated in which the medium constituents are varied to ascertain the optimum conditions producing this desired result.

' "The methods of the present invention are especially valuable in inhibiting or preventing the polyhydroxylating ability of species of Aspergillus which are capable of introducing an llu-hydroxy substituent in ll-desoiry steroids. Under the usual conditions use of these species of Aspergillus result in converting ll-desoxy steroidsto the corresponding lla-hydroxy steroids which are then further transformed by the action of the microorganism enzyme system to the corresponding 6,8,11a-dihydroxy steroid.

However in accordance with a specific embodiment of my invention, I have found that the formation of the undesired 6,8,11a-dihydroxy steroids by the action of strains of Aspergillus normally producing such dihydroxy or prevented by carefully controlling the growth of the 0X genating microorganisms. The methods by which the growth of the microorganism can be changed to produce essentially only monohydroxylation will be more readily understood by the application of such methods in hydroxylating progesterone by the action of hydroxylating'strains of Aspergillus to produce the desired lla-hydroxy progesterone in substantiallypure form and in higher yields than was heretofore possible.

Pursuant to this specific embodiment of my invention,

it is'now found that progesterone can be .hydroxylated by the action of hydroxylating strains of Aspergillus to producesubstantially only lla-hydroxy progesterone by growing such strainsin a nutrient medium deficient in a metal required for the growth of the organism, or by inhibiting the growth of the microorganism or by interference with the particular enzyme system effecting 6- hydroxylation in a medium containing a metal ion which acts as an inhibitor for the growth of the microorganism. Thus, I have found that when a strain of Aspergillus ochraceus is grown in a medium deficient in zinc ion the hydroxylating enzymes produced in such medium oxygenates progesterone to lla-hydroxy progesterone with the concomitant formation of only minor amounts of the undesired 618,1la-dihydroxy progesterones. Generally, I find; that a medium containing between about-0.0001 and 0.002 microglr'am of z'incion per ml. is sufiiciently' deficient in zinc ion to ,inhibit the enzyme system producing 65- hydroxylatiou. The fact that such small amounts of zinc will so markedly change the 6B-hydroxylation ability of Asprgillits ochrqceus'i's indeed all the more surprising and unexpected since zinc ion is, in 'fact, required for the growth of Aspergillus ochraceus.

The hydroxylation of progesterone to produce predominantly the desired llu-hydroxy, progesterone in accordance with this method of my invention is most conveniently and readily accomplished in a synthetic medium, although, if desired, this can also be accomplished in complex mediums by carefully controlling and adjusting the concentration of zinc ion present, as, for example, by removing zinc ions by the action of a chelating agent such as versene'. Alternatively, the concentration of the zinc ion in the medium can be depleted by growing another organism requiring zinc ion for its growth. Thus, when Aspergillus ochraceus is grown in a synthetic medium deficient in zinc ion the enzymes which eliect fifi-hydroxylation are not formed to any great extent. For example, when a suitable strain of Aspergillus ochraceus is grown in a medium containing 5% sucrose, 0.76% NaNO tive enzyme required for fi-hydroxylation are markedly inhibited. Thus, when Aspergillus ochraceus is grown in a synthetic medium containing sufficient amounts of zinc ion but a minor amount of cadmium ion, and progesterone is added to the growing medium, the converted product consists essentially of ll-hydroxy progesterones and only minor amountsof 613,1 la-dihydroxy progesterone is obtained. In general, I have found that this occurs when the nutrient medium contains an amount of cadmium ion equivalent to between about 0.0008 and 0.003 mg. of cadmium per ml. ofmedium. The concentration of the cadmium ion is critical and it is necessary to employ amounts within this range to obtain optimum results. Amounts less than about 0.0.008 ml. of cadmium per ml. are generally insufficient to inhibit the formation of the adaptive enzyme producing 6 3-hydroxylation and'amounts in excess of- 0.003 mg. per ml. are found to be toxic to the organism. 7 1

The hydroxylation of Progesterone-with Aspergillus ochraceus grown in the presence of cadmium ions can be effected either by adding the progesterone to the nutrient medium, or by separating the cells grown in such a medium and intimately contacting such cells with progesterone in a suitable bufier medium.

In carrying out the process of the present invention, the steroid to be hydroxylated can be added to the nutrient medium as a suspension in a suitable solvent such as water, as a solution in a solvent such as acetone and pro.- pylene glycol or in a finely divided form such as a solid micronized powder. In general, it is desirable that the steriod .be present in very finely divided form in order to permit maximum contact with the hydroxylating. en-

0.1% K HPO 0.05 MgSO .7I-I O, 0.05% KCl, 0.001%

FeSO .7'H O, and sufficient distilled water to make 100%, and progesterone is added to the medium after some growth of the microorganism has taken place, the resulting fermentation product contains essentially only 110:- hydroxy progesterones and only minor amounts of the 6B,1la-dihydroxy progesterones.

Alternatively, insteadof adding-the progesterone to the fermentation medium, the conversion of the progesterone to llu-hydroxy progesterone can be similarly efiected by intimately contacting the steroid with the enzymes formed in the zinc deficient medium. This can be accomplished by separating the cells from the medium after good growth is obtained and mixing the cellular material with the progesterone in a sodium hydroxide-potaszymes and insure completion of the reaction.

The process for effecting monohydroxylation. can be effected in both stationary and submerged cultures of the hydroxylating microorganism growing under aerobic conditions, although for practical purposes it is more conveniently carried out by growing the microorganism'under submerged conditions in a suitable fermentation me,- dium containing the steroid. The amount of steroid which can be hydroxylated will depend upon the particusium biphthalate bufier at pH 4.0, and allowing the intimately contacted mixture to incubate for a time sufficient to form the desired lla-hydroxy progesterone.

The results of these and other experiments suggest that the failure of cells grown in a zinc ion deficient medium to 6B-hydroxylate and lla-hydroxy progesterone is due to the lack of formation of the adaptive enzyme system needed to effect this secondary hydroxylation. From 'myresults, it appears that using a well balanced system in which enough zinc ion is added to allow the organism to grow, and thus bind most of the zinc ion, only a limited amount of protein synthesis occurs and the adap tive enzyme necessary for lla-hydroxylation of the progesterone is formed. .There is apparently not enough zinc ion to allow further protein synthesis and the second; adaptive enzyme necessary for ofi-hydroxylation of the progesteroh'e is not formed. While the. foregoing views express'one'possible' explanationtor the processes ofj-my'invention based upon experiments now completed, it is possible that subsequent experiments wilL.v in. fact, establish that this'explanation isin'correct. ir'a'ccordance' with a'furth'er'embo'rliinent of my invention l have found that when microorganisms are grown f certaiu metal; ionswhich inhibitthe s; we presumes er meals-e lar medium employed and the concentration of the ingredients in the medium.

The following examples illustrate. methods of carrying out this invention. a

Example 1 Ingredients Medium Medium Medium.

No. 1 No. 2 No. 3

Sucrose -percent-.. 5 6 5 NHZNO: (in 5 NaNO: d0' 0; 76 0. 76 KgHPO -do---- 0. 0. 1 0. 1 MgSO4.7H;0. .40.... 0.5 0.05 1 0.05 1- do 0.05 0.05 FeCl3.6HgO- do;- 0. 008 FBSO4.7H10.- "do"-.. 0, 001 0. 001 2115 04.71110; --do.- 0. (105 Distilled water. to do- 100 100 100 zns0,. 7H,o mi! 11.. so FQSQ4-7H2O mgJL- 80 MediumsNo. 1 and 2 wereprepared by dissolving the I ingredients in the amounts shown in distilled water.

Medium 3. was made up the same as No. 2' and then supplemented by theaddition of 50 mg. of ZnSO4.7H O and mgs. of FeSO .7H O per, liter oiNo. 2 medium.

50 ml. portions of the ahovemediumswerethen placed in 250. ml. Erlenmeyerflasksand sterilizedby autoclaving at 15.p.s.i.g. for 17 minutes... The. flasks were then inoculated' with a small amount of a. vegetative: growth of Aspergillus ochraceus NRRL-450 strain 260-4118 and the flasks, incubatedat. 28 C. on rotary shakers moving at 220 r.p.m. and describing a circle, of 1.5 inches in diameter.:-After 24-48 hours of incubation, a solution of 10 g. gof progesteronein 2,5 ml. of propylene glycolwas addrsl'tq w of t e ta After the addition of the LOOOOOO ample supple- 5, 10, 15

the sample conlla-hydroxy progesterone.

was added and The Percent conversion con to 110:- hydroxyprogesterone nd mg. of progesterone glycol was added. After r for 12 hours, the cells 0 strain 2604718 d in Example 1. va

ayed in accorda mple 1, and the results Progesterone, percent 111100111- verted .i i i n nnu mmwh m... o

propylene na yze or residual st Employing the Zatfaroni tec 1, it was found that 6 3, lla-dihydroxy Example 4 propylene glycol shaker continued. re then ass Example 5 prepared as described in Ex medium were then further .8H O at the levels of 1,

hthalatebufier at pH 4.0. The cells were ed in fresh buffer a ssolved in 2.5 ml. of

er incubation on the shake unchanged progesteron Medium No. 2 was prepared, sterilized, inoculated with spergillus ochraceus NRRL incubated for 4 days as describe 15 end of this incubation period potassium bip then suspend di fiurth d the three 5 and buifer were a l d f conversion products. described in Example tained 4.1% progesterone and 11.2%

gesterone dissolved in the incubation on the fermented products we the procedures described in Exa 2 summarized in the following table:

ryness at room en up in an apquots spotted atograms were nd method of 10 h pure methanol, products determined e progesterone to alculated from the above, the following ved at intervals for analysis. ole broth was homogenized minute, then extracted three ree mediums supple- For assay, the m1. of wh in a Waring blender for one times with 25 ml. amounts of chloroform an extracts combined and taken down to d temperature. The dried extract was tak propriate amount of pure methanol and ali paper. The chrom ing the solvent systems a The conversion products were located by means of an ultraviolet light scanner, then eluted wit andthe optical densities of the eluted at 2400 A. The percent conversion of th on products was then c progesterone, the flasks were replaced on'the shaker and individual flasks were remo on Wattman No. 1 filter developed us Zaifaroni.

the transformati optical densities.

' Using the procedures described results were obtained with the th mented with progesterone:

Medium No. 2 was Portions of this mented with CdSO Percent;

to 65, 11adihydroxyprogesterone Percent conversion conversion to 11:1- hydroxyprogesterone Progesteronc,

Percent remaining Age of samples, hrs.

uAan m .l em 05 t v t re m mfm owmeer mmm n mmm mm an Wm a smw .dle 1 PS AL 11 cm 0.. o o mum mn wmflk uewu y l SM m ma fm mm mw m ew w w e u 1 d ac -1 5. e ew I. k 6 v n f .W. umfie sm o m m %W m m 0 S fmuw wa h meew mm I .OL mhe mm. rwmf. measm vl ea n f c r e .nr mu aw hnnm. 1P oNmm mmham osm S e m c mam m m H mmm P mmw mh mm dw mm mm nam mm m w r mm mm m mmmmmo wt mmEpm sta. lwrad M m "w 8713 g -t m ne mm mm QM W5 S e sma w woww m d 1d p 7 h .2 w a m m m emm" m 0mm e .mr m 3P m mm m 5 m mnm a.. iama o S 1 mi e u r m m. d4 m e m a m dnw y v ..R .3 mm M m t 1 s mmm wm .11 w @1 m w .mWt 2MP e W W omm mm w E m S fd N 0 m n m m s m d mm w mm mm n mw 16m w mem ME AS&

The results of analysiswere tabulated asifollowsr' Percent 'Pereent. v Age of liroges-v conversion conversion Levelot3CdSO .8H O Sample, terone, to a to 613,111-

hrs. percent hydroxy dihydroxyremaining progesprogesterone terone v 6 34.2 64.3 5.5 12' 6.4 5611" 13.3 1mg./l 24. 12.6 32.2 32.8 48 '0 12.1 49.9 72 7.5 39.6 1 96- (I 13.8 45.7 6 27.6.. 21.7,. 5.3 12 3.2 49.5 17.0 sing/l. 24- 10. 2 9.1- 42.1 L 48 I 0 6.2' 48.7 72 0 7.9 42.5 96 0' 80.0 42.3 6 63.2 10.5 4.4 12 32.3 32.1 0 10 mg.[l 24 9.8 64.1 8.4 48 2.1 46.0. 16.7 72 11.1 76.8 i 4.7 96 0 i 6.1 I 54.8

6 12 59.0 16.7 15 mg./l 24 39. 6 59.3 6.1 48 7.5 61.2 5.3 72 4.5 46.8 14.1 96' mg.ll No growth Various changes and modifications may be made in my invention, certain preferred embodiments of which are herein disclosed, without, departing from the scope thereof; to the extent that these changes and modifications are within the. scope of the appended claims, they are to be considered as part of my invention.

Iclaim:

1. The process for the production of improved hydroxylating enzymes capable of converting ll-desoxy steroids to lla-hydroxy steroids and inhibiting the concomitant formation of polyhydroxylated steroids, which comprises growing a hydroxylating strain of Aspergillus in a medium containing from about 0.001 to about 0.002 micrograms of zinc ion per ml.

2. The process of claim 1 wherein the hydroxylating strain of Aspergillus'is Aspergillus ochraceus.

3. The process for the production of improved hydroxylating enzymes capable of converting ll-desoxy steroids to l'lccvhydroxy steroids and inhibiting thecon-- comitant formation of polyhyd'roxylated steroids, which comprises growing a' hydroxylating ,strain' 'of Aspergillus in a medium containing an amount of cadmium equivalent tofrom about 0.0008 to about 0.003 mg. of cadmium per ml.

4. The process of claim 3 wherein the hydroxylating strain of Aspergillus is Aspergillus ochraceus. I

5. In the process for the conversion of an ll-desoxy steroid to an l-l'u-hydroxy steroid by intimately contacting said ll-desoxy steroid with hydroxylating enzymes produced by growing a hydroxylating strain of Aspergillus, the improvement which comprises growing said hydroxylating strain of Aspergillus in a' medium containing from about 0.001 to about 0.002 micrograms of zinc ion per ml. 7 i I 6. The process of claim 5 wherein the hydroxyl'ating strain of Aspergillus is Aspergillus achraceus. I

7. In the process for the conversion of an ll-desoxy steroid to an lla-hydroxy steroid by intimately contacting said ll-desoxy steroid with hydroxylating enzymes produced by growing a hydroxylating strain of Aspergillus, the improvement which comprises growing said hydroxylatingstrain of Aspergillus in a medium containing a soluble cadmium salt having an amount of cadmium equivalent to from about 0.0008 to about 0.003 mg. of cadmium per ml.

8'. The process of claim 7 wherein the hydroxylating strain of Aspergillus is Aspergillus ochraceus.

References Cited in the file of this patent UNITED STATES PATENTS Murray July 8, 1952 Murray Aug. 18, 1953 OTHER REFERENCES 

1. THE PROCESS FOR THE PRODUCTION OF IMPROVED HYDROXYLATING ENZYMES CAPABLE OF CONVERTING 11-DESOXY STEROIDS TO 11A-HYDROXY STEROIDS AND INHIBITING THE CONCOMITANT FORMATION OF POLYHYDOXYLATED STEROIDS, WHICH COMPRISES GROWING A HYDROXYLATING STRAIN OF ASPERGILLUS IN A MEDIUM CONTAINING FROM 0.001 TO ABOUT 0.002 MICROGRAMS OF ZINC ION PER ML. 